One of the critical determiners of oxygen delivery to brain is diffusion from the vascular system. This is controlled by open capillary density, surface area and diameter of the perfused capillary bed of all brain regions. While regional blood flow, the other determiner, can easily be measured, until now open capillary density and capillary reserves could not be quantitatively determined regionally in brain. Our hypotheses are that capillary reserves are one of the key unexplored mechanisms available to different brain regions to protect them against 02 supply deficits and that different regions have different reserves. To test these hypotheses, we have developed a new method to determine perfused and total capillary density on a regional basis in brain. A fluorescent dye is injected into the vein of a rat. After an appropriate time, the head is removed and quick-frozen. Photomicrographs of thin sections of various brain regions are obtained, where the fluorescence shows the perfused vessels. The tissue is stained and rephotographed to reveal the total capillary network. Morphometric analysis is used to determine the parameters of interest in the total and perfused capillary networks. The method has been extensively validated in brain, muscle and omentum to show that the technique does not alter the capillary network, that both the total and perfused vessels are fully visualized and that there is little tissue shrinkage. This technique will be applied to study the alterations in the perfused capillary network of conscious Sprague Dawley rats. Prior surgery will be done to catheterize a femoral artery and vein. These animals will be used to study the effect of reductions in brain 02 supply. 02 supply will be reduced by hypoxia, hypocapnia, anemia, carbon monoxide, hemorrhage and stroke. This will provide new information on how the brain handles reductions in 02 supply on a regional basis in terms of alterations in diffusion distances. This new method allows study of the functional state of the capillary bed in normal, stressful and disease conditions regionally within an organ like brain and thus will allow a better understanding of the control of the delivery of substances from blood to cell.